Laser induced isolation and cultivation of single microbial cells

Abstract

Single cell isolation and cultivation play an important role in studying physiology, gene expression and functions of microorganisms. Laser Induced Forward Transfer Technique (LIFT) has been applied to isolate single cells but the cell viability after sorting is unclear. We demonstrate that a three-layer LIFT system could be applied to isolate single cells of Gram-negative (E. coli), Gram-positive (Lactobacillus rhamnosus GG, LGG), and eukaryotic microorganisms (Saccharomyces cerevisiae) and the sorted single cells were able to be cultured. The experiment results showed that the average cultivation recovery rate of the ejected single cells were 58% for Saccharomyces cerevisiae, 22% for E. coli, and 74% for Lactobacillus rhamnosus GG (LGG). The identities of the cultured cells from single cell sorting were confirmed by using colony PCR with 16S-rRNA for bacteria and large unit rRNA for yeast and subsequent sequencing. This precise sorting and cultivation technique of live single microbial cells can be coupled with other microscopic approaches (e.g. fluorescent and Raman microscopy) to culture single microorganisms with specific functions, revealing their roles in the natural community.ImportanceSingle cell isolation and cultivation are crucial to recover microorganisms for the study of physiology, gene expression and functions. We developed a laser induced cell sorting technology to precisely isolate single microbial cells from a microscopic slide. More importantly, the isolated single microbial cells are still viable for cultivation. We demonstrate to apply the live sorting method to isolate and cultivate single cells of Gram-negative (E. coli), Gram-positive (Lactobacillus rhamnosus GG, LGG), and eukaryotic microorganisms (Saccharomyces cerevisiae). This precise sorting and cultivation technique can be coupled with other microscopic approaches (e.g. fluorescent and Raman microscopy) to culture specifically targeted single microorganisms from microbial community.Abstract Graphic

Single cell sorting of young yeast based on label‐free fluorescence lifetime imaging microscopy

“…In this work, upon 95% yeast in control group can survive, which could be explained by the fact that the yeast cells were in a sterile and humid environment during the entire operation process. Moreover, lower laser energy for sorting was used than Liu et al reported [21], Liu et al reported 58% S. cerevisiae survived after sorting by LIFT, which was much lower than the result of 84% ± 3% in this work. The low damage to target cells of the present method favors the sorting of single cell and other subsequent single-cell research work.…”

“…Liang et al had sorted three microorganisms including S. cerevisiae, Escherichia coli, and Lactobacillus rhamnosus GG by a three-layer LIFT system. The average re-cultivation ratio reached 58%, 22%, and 74%, respectively [21]. It made it possible to sort target microorganisms accurately, though the average re-cultivation rate still needed to promote.…”